Recent patch clamp experiments on mast cells undergoing exocytosis have demonstrated the properties of the single regulated fusion event at a level of detail comparable to that of single ion channel recording. Dr. Fernandez will make use of this assay system to examine the role and mode of action of the mast cell homologs of the synaptic vesicle membrane proteins. He will use PCR-cloning to identify the mast cell homologs of rab-3A, rabphilin-3A and other synaptic membrane proteins. He will attempt to determine the roles of these proteins in regulating exocytic fusion using protein derived agents in combination with patch clamp and imaging measurements. Initial work will focus on rab3 and its putative effector proteins. A ubiquitous stimulus for exocytotic vesicle fusion is an elevation of intracellular calcium. After defining what constitutes a calcium stimulus, Dr. Fernandez will study the interaction between rab3 proteins and a calcium signal to cause exocytosis. Recent data suggest that the calcium signal that is effective in triggering exocytosis is a gradient localized near the plasma membrane, rather than a homogeneous rise in intracellular calcium. Using a novel, submicrosecond calcium imaging microscope, he will determine what constitutes an effective calcium stimulus in mast cells. These studies should illuminate the mechanisms by which intracellular signals generated by rab proteins and calcium gradients regulate the activity of effector proteins to cause exocytosis. He will extend these studies to other secretory vesicle proteins in an effort to understand the relationship between the signalling pathways and exocytotic vesicle fusion.